Gabriele Gille

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinsons disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Environmental toxins trigger PD-like progression via increased alpha-synuclein release from enteric neurons in mice

Pathological studies on Parkinsons disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate.

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a well known and popular herbal medicine used worldwide. Among more than 30 ginsenosides, the active ingredients of ginseng, ginsenosides Rb1 and Rg1 are regarded as the main compounds responsible for many pharmaceutical actions of ginseng. In our study, primary cultures from embryonic mouse mesencephala were exposed to neurotoxic glutamate concentration and potential protective effects of these two ginsenosides on survival and neuritic growth of dopaminergic cells were tested. Treatment of primary mesencephalic culture with 500 microM glutamate for 15 min on the 10th day in vitro (DIV) increased the release of lactate dehydrogenase (LDH) into the culture medium, the propidium iodide (PI) uptake by cultured cells and the total number of nuclei with condensed and fragmented chromatin (apoptotic features) as evaluated with Hoechst 33342. Moreover, it extensively decreased the number of tyrosine hydroxylase immunopositive (TH+) cells and adversely affected the length and number of their neuronal processes. The toxic effect of glutamate was primarily mediated by over-activation of N-methyl-D-aspartate receptor (NMDA) as treatment of cultured cells with (+)-MK 801, an NMDA receptor antagonist, nearly abolished dopaminergic cells loss and LDH release induced by glutamate. When either ginsenoside was added alone for six consecutive days (at final concentrations 0.1, 1, 10, 20 microM), ginsenoside Rb1 (at 10 microM) significantly enhanced the survival of dopaminergic neurons compared to untreated controls. In these cultures, neurite lengths and numbers were not affected by both ginsenosides. Against glutamate exposure, ginsenosides Rb1 and Rg1 could not prevent cell death. However when pre-treating for 4 days or post-treating for 2 days following glutamate exposure, they significantly increased the numbers and lengths of neurites of surviving dopaminergic cells. Thus our study indicates that ginsenosides Rb1 and Rg1 have a partial neurotrophic and neuroprotective role in dopaminergic cell culture.

Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration

Although the definite etiology of Parkinsons disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinsons disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinsons disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

Ginsenosides Rb1 and Rg1 effects on survival and neurite growth of MPP+-affected mesencephalic dopaminergic cells

Summary.Ginsenosides Rb1 and Rg1 are the main active ingredients of Panax ginseng C.A. Meyer (Araliaceae). They appear to exert protection against ischaemia and anoxic damage in animal models, suggesting an antioxidative and cytoprotective role. In our study, primary cultures from embryonic mouse mesencephalon are applied to examine the effects of these two ginsenosides on neuritic growth of dopaminergic cells and their survival affected by 1-methyl-4-phenylpyridinium-iodide (MPP+). Ginsenoside Rb1 (at 10 µM) enhanced the survival of dopaminergic neurons by 19% compared to untreated control. MPP+ (at 1 µM) significantly reduced the number of dopaminergic neurons and severely affected neuronal processes. Both ginsenosides counteracted these degenerations and significantly protected lengths and numbers of neurites of TH+ cells. Both compounds however could not prevent the cell loss caused by MPP+. Our study thus indicates partial neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 in dopaminergic cell culture.

Iron-dependent functions of mitochondria—relation to neurodegeneration

A number of neurodegenerative diseases are associated with iron dyshomeostasis and mitochondrial dysfunction. However, the pathomechanistic interplay between iron and mitochondria varies. This review summarises the physiological role of iron in mitochondria and subsequently exemplifies two neurodegenerative diseases with disturbed iron function in mitochondria: inherited Friedreich ataxia (FRDA) and idiopathic Parkinson disease (PD). In eukaryotes, mitochondria are main consumers of iron. The respiratory chain relies on iron-containing redox systems in the form of complexes I–III with iron–sulphur clusters and cytochromes with haem as prosthetic groups. The bifunctional enzyme aconitase is not only important in the citric acid cycle, but also functions as a key regulator of cell iron metabolism. Haem biosynthesis occurs partially in mitochondria as well as the biogenesis of iron–sulphur clusters that are co-factors in numerous iron–sulphur proteins. FRDA is characterised by a mutation of the frataxin gene, the protein of which serves as an iron chaperone in iron–sulphur cluster assembly. The lack of frataxin expression leads to defective iron–sulphur cluster biogenesis with decreased respiratory and aconitase activity. The resulting mitochondrial iron overload might fuel reactive oxygen species formation and contribute to clinical signs of oxidative stress. PD is typically associated with an increased iron content of the substantia nigra, the causes of which are largely unknown. Recent research demonstrated raised iron levels in individual dopaminergic neurons of the substantia nigra. Moreover, transferrin/transferrin receptor 2 mediated transport of iron into the mitochondria of these neurons was identified together with increased transferrin immunoreactivity. Resulting accumulation of iron into mitochondria might lead to oxidative stress damaging iron–sulphur cluster-containing proteins.

Oxidative Stress to Dopaminergic Neurons as Models of Parkinson's Disease

Abstract: The effects of exogenous toxins (MPP+, rotenone) and potentially neurotoxic properties of levodopa (L‐DOPA) on the survival rate of dopaminergic neurons in dissociated primary culture are presented. Dopamine agonists show a capacity to counteract MPP+‐toxicity. Moreover, a preserving potential of the antioxidant and bioenergetic coenzyme Q10 (CoQ10) on the activities of tyrosine hydroxylase (TH), complexes I and II of the respiratory chain, and hexokinase activity in striatal slice cultures against MPP+ is demonstrated.

Neurotoxic mechanisms of 2,9‐dimethyl‐β‐carbolinium ion in primary dopaminergic culture

β‐Carbolines are potential endogenous and exogenous neurotoxicants that may contribute to the pathogenesis of Parkinsons disease. The 2,9‐dimethyl‐β‐carbolinium ion (either 2,9‐dimethyl‐β‐norharmanium or 2,9‐Me2NH+) was found to be neurotoxic in primary mesencephalic cultures and to be a potent inhibitor of mitochondrial complex I. However, the precise mechanisms of cell death remained obscure. Here, we investigated the mechanism of cell death in primary dopaminergic cultures of the mouse mesencephalon mediated by 2,9‐Me2NH+. The β‐carboline caused preferential death of dopaminergic neurones, which could not be attributed to cellular uptake via the dopamine transporter. Transient incubation with 2,9‐Me2NH+ for 48 h caused a progressive deterioration in the morphology of dopaminergic neurones during a 5‐day recovery period and persistent damage to the overall culture. An increase in free radical production and caspase‐3 activity, as well as a decrease of respiratory activity, mitochondrial membrane potential and ATP content, contributed to toxicity and pointed to an apoptotic mode of cell death, although a significant quantity of cells dying via necrosis were present simultaneously. These data underline the preferential susceptibility of dopaminergic neurones to 2,9‐Me2NH+ as a potent, oxidative stress generating neurotoxin.

Pergolide protects dopaminergic neurons in primary culture under stress conditions

Summary. Dopamine agonists are an important therapeutic strategy in the treatment of Parkinsons disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D2 agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 μM. About 100 fold higher concentrations (0.1 μM) were necessary to partially reverse the toxic effects of 10 μM 1-methyl-4-phenylpyridinium (MPP+). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 μM L-DOPA. Furthermore, between 0.001–0.1 μM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress.

9-Methyl-β-carboline up-regulates the appearance of differentiated dopaminergic neurones in primary mesencephalic culture

beta-Carbolines (BCs) derive from tryptophan and its derivatives. They are formed endogenously in humans and mammals and occur inter alia in cooked meat and tobacco smoke. They have been detected in human brain, cerebrospinal fluid, and plasma. Up to now they were predominantly identified as compounds exhibiting neurotoxic actions. Since significantly higher amounts are present in parkinsonian patients, they are regarded as potential pathogenetic factors in Parkinsons disease. We identified for the first time a BC (9-methyl-BC; 9-me-BC) exerting neuroprotective and neuron-differentiating effects. Treatment of primary mesencephalic dopaminergic cultures with 9-me-BC inhibited the basal release of lactate dehydrogenase and reduced the number of cells stained with propidium iodide. Caspase-3 activity was decreased, the total protein content was unchanged and ATP content was increased. Furthermore, the expression of inflammation-related genes was reduced. The number of differentiated dopaminergic neurones was significantly increased and a wide array of neurotrophic/transcription factors (Shh, Wnt1, Wnt5a, En1, En2, Nurr1, Pitx3) and marker genes (Th, Dat, Aldh1a1) decisive for dopaminergic differentiation was stimulated. Consistently, the dopamine content was slightly, although non-significantly, increased and the dopamine uptake capacity was elevated. An anti-proliferative effect was observed in human neuroblastoma SH-SY5Y cells which is consistent with a reduced incorporation of bromodesoxyuridine into the DNA of primary mesencephalic cells. Whether the additional dopaminergic neurones in primary culture derive from dopaminergic precursor cells, previously tyrosine hydroxylase negative dopaminergic neurones or are the result of a transdifferentiation process remains to be established.